Ongoing studies in this project concern the cell biology, biochemistry and molecular biology of Leishmania, a group of protozoan pathogens of humans. All Leishmania parasites undergo a dimorphic life cycle: 1) in mammals (humans), they multiply as obligate intracellular forms [amastigotes] within the lysosomal system of macrophages, eventually destroying these cells and 2) within their insect vectors (blood-sucking sandflies), they differentiate and multiply as, extracellular forms [promastigotes] within the alimentary tract and eventually migrate to the mouth parts for transmission. By World Health Organization estimates, Leishmania parasites annually cause well-over 12 million cases of human disease (leishmaniasis) worldwide. In infected humans, these parasites destroy macrophages within the skin or internal organs (i.e. spleen, liver and bone marrow) causing either large and disfiguring, malignant skin ulcers (e.g. caused by L. mexicana) or degenerative and most often fatal visceral disease (e.g. caused by L. donovani). Previous studies from our laboratory have established that Leishmania parasites constitutively secrete over 40 different soluble protein, glycoprotein and carbohydrate constituents. Such secretory products can readily permeate throughout and presumably alter the host micro-environments in which Leishmania reside. Thus, an understanding of the nature of these parasite products seems essential. To that end, several parasite secretory enzymes and regulatory proteins are being investigated toward defining their functional roles in the survival, maintenance, growth and transmission of these organisms. Further, genes encoding these proteins have been identified and characterized for the first time toward defining their expression and regulation during parasite growth, development and differentiation. For example, during the past year, our studies have elucidated the enzymatic activity, gene structure and chromosomal locus of the L. mexicana chitinase. Further, using various chimeric constructs, we showed that during parasite development and differentiation, the expression of this enzyme is regulated at the post-transcriptional level. In addition, the genes for the several unique L. donovani histidine secretory- (LdSAcPs) and membrane- (LdMAcP) acid phosphatases were used as probes in molecular studies. Results of those biochemical and molecular studies showed that this family of enzymes was functionally conserved among all pathogenic leishmanial species examined as well as, other more distantly related trypanosomatid parasites of humans. The conservation of such LdSAcPs and LdMAcP homologs amongst all pathogenic Leishmania sps. suggests that they must play significant functional roles in the growth, development and survival of all members of this important group of human pathogens. Moreover, we made chimeric constructs of several of these genes in tandem with the green fluorescent protein (-GFP) to determine the unique molecular signals responsible for targeting these enzymes into and through the parasite secretory pathway. Further, our studies involving gene-deletion, -mutation, and -over expression of the L. donovani calreticulin demonstrated that this resident ER chaperone-folding protein is in fact, essential to the survival of these human pathogens. In addition, using GFP constructs of several different truncated surface membrane proteins, we defined a unique endosomal trafficking pathway in Leishmania which interfaces with the secretory pathway and functions, at least in part, to regulate protein over-expression of these organisms.